Project Summary/Abstract: H2O2 is the most important ROS involved in cell signaling in the lung. PMA has been used to study H2O2-mediated signaling, but asbestosis is a good model to explore the role of H2O2 in cell signaling because asbestos is biologically relevant and is known to induce high levels of H2O2. The release of TNF-alpha by alveolar macrophages in asbestosis patients is critical in the development of the disease; however, the mechanism linking H2O2 generation to TNF-alpha production is not known. Another feature of asbestosis is that alveolar macrophages from patients resemble monocytes. Our data show that monocytes produce TNF-alpha when stimulated, in vitro, with asbestos unlike alveolar macrophages. Thus, human monocytes provide a good model to explore TNF-alpha gene expression, as it relates to H2O2 generation. In this regard, our preliminary data show that H2O2 is necessary for optimal production of TNF-alpha in monocytes and that the source of H2O2 is primarily from Cu,Zn-SOD. Our preliminary data also demonstrate that Cu,Zn-SOD is highly expressed in alveolar macrophages obtained from patients with asbestosis. Since our data shows that alveolar macrophages from asbestosis patients have a high level p38 MAP kinase activity and absent ERK activity and that H2O2 activates p38 and inhibits ERK, we hypothesize that H2O2 plays a pivotal role in differentially modulating MAP kinase activity, and Cu,Zn-SOD enhances differential MAP kinase activation and TNF-alpha production in human monocytes. We will use PMA in the initial studies to understand the role of H2O2 in cell signaling in addition to performing studies using asbestos, which is more biologically relevant. In Aim 1 we will determine if Cu,Zn-SOD-induced H2O2 modulates MAP kinase activation. Since MAP kinase activity is often controlled by phosphatases, we will investigate if H2O2 is oxidizing a phosphatase, MKP-1, and inducing expression of PP2A, which results in p38 activation and ERK inactivation, respectively. The comparison of H2O2 generation and MAP activation in wild-type and Cu,Zn- SOD KO mice will provide biological relevance. In Aim 2 we will determine if Cu,Zn-SOD-induced H2O2 increases TNF-alpha production in monocytes and if optimal TNF-alpha expression depends on differential MAP kinase activity. We will compare MAP kinase and Cu,Zn-SOD activity and H2O2 generation in alveolar macrophages obtained from asbestosis patients to normal macrophages. Additional biological relevance will be provided by investigating the development of interstitial fibrosis in wild-type and Cu,Zn- SOD KO mice. Project Narrative: Exposure to asbestos and other environmental agents, such as diesel exhaust and particulate matter, increase hydrogen peroxide generation in the lung. The goal of the project is to understand the mechanisms by which hydrogen peroxide causes lung injury and fibrosis. Although the studies in this proposal relate to asbestosis, they will also apply to other inflammatory and fibrotic lung diseases.